2.1 Poly-β-1→4-N-acetylglucosamine Polymers (pGlcNAc) and Other Polysaccharides
Poly-β-1→4-N-acetylglucosamine polysaccharide species are typically polymers of high molecular weight whose constituent monosaccharide sugars are attached in a 1→4 conformation. There is a body of literature on the properties, activities, and uses of polysaccharides that consist, in part, of pGlcNAc. A class of such materials has been generically referred to as “chitin”, while deacetylated chitin derivatives have been referred to as “chitosan”. When these terms were first used, around 1823, it was believed that chitin and chitosan always occurred in nature as distinct, well-defined, unique, and invariant chemical species, with chitin being fully acetylated and chitosan being fully deacetylated compositions. It was approximately a century later, however, before it was discovered that the terms “chitin” and “chitosan” are, in fact, very ambiguous. Rather than referring to well-defined compounds, these terms actually refer to a family of compounds that exhibit widely differing physical and chemical properties. These differences are due to the products' varying molecular weights, varying degrees of acetylation, and the presence of contaminants such as covalently bound, species-specific proteins, single amino acid and inorganic contaminants. Even today, the terms “chitin” and “chitosan” are used ambiguously, and actually refer to poorly defined mixtures of many different compounds.
For example, the properties of “chitins” isolated from conventional sources such as crustacean outer shells and fungal mycelial mats are unpredictably variable. Such variations are due not only to species differences but are also due to varying environmental and seasonal effects that determine some of the biochemical characteristics of the “chitin”-producing species. In fact, the unpredictable variability of raw material is largely responsible for the slow growth of chitin-based industries.
Production of pGlcNAc, that is either fully acetylated or uncontaminated by organic or inorganic impurities is challenging. While McLachlan et al. (McLachlan, A. G. et al., 1965, Can. J. Botany 43:707-713) reported the isolation of chitin, subsequent studies have shown that the “pure” substance obtained, in fact contained proteins and other contaminants.
Deacetylated and partially deacetylated chitin preparations exhibit potentially beneficial chemical properties, such as high reactivity, dense cationic charges, powerful metal chelating capacity, the ability to covalently attach proteins, and solubility in many aqueous solvents. The unpredictable variability of these preparations, as described above, however, severely limits the utility of these heterogenous compounds. For example, the currently available “chitins” and “chitosans” give rise to irreproducible data and to unacceptably wide variations in experimental results. Additionally, the available preparations are not sufficiently homogenous or pure, and the preparation constituents are not sufficiently reproducible for these preparations to be acceptable for use in applications, especially in medical ones.
Certain studies have met with success in producing a pure and consistent poly-β-1-4→N-acetylglucosamine product useful in therapeutic applications (Kulling et al., 1998, Endoscopy 30(3):S41-2; Cole et al., 1997, Clin. Cancer Res. 3(6):867-73; Maitre et al., 1999, Clin. Cancer Res. 1999, 5(5):1173-82). Uses of poly-β-1-4→N-acetylglucosamine derivatives as hemostatic agents or wound healing agents have been shown to be particularly effective (Cole et al. 1999. Surgery. 126(3):510-7; Chan et al., 2000, J. Trauma 48(3):454-7). In addition, several patents relating to uses and derivatives of poly-β-1-4→N-acetylglucosamine products exist. U.S. Pat. No. 5,622,834 describes chemical and mechanical force methods for isolating poly-β-1-4→N-acetylglucosamine of about 4,000 to 150,000 subunits that is free of protein, substantially free of other organic contaminants, and substantially free of inorganic contaminants. U.S. Pat. No. 5,623,064 describes poly-β-1-4→N-acetylglucosamine and derivatives thereof having varying degrees of purity, acetylation, and molecular weight. U.S. Pat. No. 5,846,952 describes drug/poly-β-1-4→N-acetylglucosamine compositions. U.S. Pat. No. 5,624,679 describes biodegradable barrier-forming material comprising poly-β-1-4→acetylglucosamine or a derivative thereof. U.S. Pat. No. 5,858,350 describes a biological cell encapsulated by poly-β-1-4→N-acetylglucosamine or a derivative thereof. U.S. Pat. No. 5,635,493 describes anti-tumor drug/poly-β-1-4→N-acetylglucosamine, including a drug encapsulated by poly-β-1-4→N-acetylglucosamine and methods for anti-tumor delivery of such drugs. U.S. Pat. No. 5,686,115 describes hybrid compositions comprising poly-β-1-4→N-acetylglucosamine derivative crosslinked to another compound. U.S. Pat. No. 6,063,911 describes anti-tumor compositions comprising endothelin antagonists and poly-β-1-4→N-acetylglucosamine or poly-β-1-4→N-acetylglucosamine derivatives. U.S. Pat. No. 5,510,102 discloses compositions that act as coagulants and may be used to promote clotting of a wound by placing the compositions in contact with the wound where the composition comprises platelet rich plasma plus a biocompatable polymer that is a hemostatic agent such as alginate.
2.2 Platelet Interactions
The compositions of the invention can be used in a variety of ways to preserve platelets for longer periods of time because platelets that can be activated or remain activated after storage can produce many beneficial compounds and interactions Viability of stored platelets also has important implications in autologous therapies or procedures where a subject is administered compositions of the invention comprising self-derived platelets that have been stored.
Investigation of the interaction of platelets with “foreign” materials that has been ongoing since the original discovery of the platelet (Donne, 1842, Bizzozero et al., 1882a and 1882b). Existing evidence indicates that the interaction of platelets with foreign materials involves two steps. First, serum proteins (Silberberg, 1962, J. Physical Chem. 66:1872-1883), including adhesion proteins such as fibrinogen (Mason et al., 1971, Proc. Soc. Exp. Biol. Med. 320:123-128; Zucker and Vromen, 1969, Proc. Nat. Acad. Sci. U.S.A. 87:758-762; Feuerstein and Sheppard, 1993, Biomaterials 14:137-147), fibronectin (Lewandowska et al., 1992, J. Biomedical Materials Res. 26:1343-1363) and von Willebrand factor (vWF) (Kuwahara et al., 1999, Blood 94:1149-1155) change conformation when absorbed onto surfaces. These interactions involve multiple points of attachment of the macromolecules to the foreign materials that are “random” in nature, but the result is an alteration in solution-phase protein conformation. Secondly, the altered conformations of the absorbed proteins expose structural domains that “activate” platelet cell surface proteins. For example, fibrinogen might assume a “fibrin-like” conformation, and thus initiate outside-in signaling through the alpha2bbeta3 complex. Similarly, absorbed vWF might assume a “sheared” conformation and bind to GPIb-IX complexes. A substantial body of evidence indeed indicates that alpha2bbeta3 (e.g., Rozenberg and Stormorken, 1967, Scand. J. Clin. Lab. Invest. 19:82-85; Coller et al., 1983, J. Clin. Invest. 72:325-338; Ginsberg et al., 1983, J. Clin. Invest. 71:619-624) and GPIb-IX (e.g., Mattson et al., 1984, Scanning Electron Microscopy 4:1941-1950) are important for the interaction of platelets with absorbed plasma proteins. While the processes through which alpha2bbeta3, GPIb-IX and other surface proteins are activated by absorbed ligands are largely undefined, it is likely that high local levels of the absorbed plasma proteins cluster glycoprotein cell surface proteins on the platelet membrane to organize cytoskeletal signaling machinery for active outside-in signaling complexes.
The plasma protein FXII (Hageman factor) has been shown to be linked with the plasma defense systems of coagulation, fibrinolysis, kallikrein-kinin and complement. FXII can be activated by complex phemonenon involving negatively charged surfaces. FXII, also binds and proteolytically activates upon contact with many anionic surfaces, and is peripherally associated (Iatridis et al., 1953, Thromb. Et Diath. 11:355-371) with the platelet surface for activation of the intrinsic coagulation pathway in the microenvironment of the cell. While the series of proteolytic steps that occurs on the platelet surface are poorly understood, the protective effect of aprotinin on platelets during cardiopulmonary bypass has been hypothesized to in part originate from an inhibition of “kallikrein-like” proteolytic events as the cells contact foreign surfaces in the extracorpealizaton device (Bradfield and Bode, 2002, Blood, in press).
Intracellular calcium signaling plays a central role in orchestrating the platelet activation response, and has been shown to occur when platelets contact glass (Ikeda et al., 1996, J. Cellular Biocem. 61:292-3000; Hussain and Mahaut-Smith, 1999, J. Physiol. 524:713-718) and polylysine (Ikeda et al., 1996, J. Cellular Biocem. 61:292-3000). The known mechanisms by which platelets interact with substances remain elusive.
2.3 Problems with Blood Storage
Concern has been steadily growing over both the national, and worldwide blood supplies. Both the integrity and reliability of existing supplies, and the ability to build larger stocks over time, have been brought into question. One reason for this is the relatively short period of storage stability of blood products. Currently, packed RBCs (red blood cell concentrates, or RCC), the dominant form of blood product for transfusions and the like, are limited to a 42-day storage period. After that time, ATP levels fall substantially, coupled with a significant loss of pH, strongly indicating a lack of viability, or, if viable, an extremely short circulation life upon infusion, in vivo. Whole blood is not stored for substantial periods. For platelets, the current storage period is even shorter, with the standard being 5 days at 22° C.
Typically, platelets are stored at 20-24° C. (approximately room temperature) with continuous gentle agitation. They are typically stored for up to 5 days, after which time they have to be discarded. Red blood cells are generally stored for 42 days. There exists a need in the art to preserve platelets and red blood cells for longer periods of time. For example, according to the congressional report entitled America's Blood Supply in the Aftermath of Sep. 11, 2001, following the Sep. 11, 2001 attacks in New York, about half a million more units of blood were collected that normally were for that time of year (H.R. Comm. Energy and Commerce, America's Blood Supply in the Aftermath of Sep. 11, 2001, 107th Cong., 107-137 (Sep. 10, 2002)). The report also indicates that large portions of the blood collected during this period was disposed of because the blood supply system could not process the blood fast enough or store the blood for extended periods of time. Donors were discouraged to learn the fate of their blood and those in the blood supply community were also discouraged by their inability to handle the large volume of blood, once it became evident that the low number of survivors meant a limited need for emergency blood transfusions. The need for donated blood, and thus improved storage of donated blood, is still critical. Every 3 seconds a patient in the U.S. requires blood, yet blood is a human tissue that cannot be manufactured, it must be donated.
The compositions of the invention comprising platelets can be used in a variety of therapeutic applications, and are particularly advantageous over present applications in that they make use of stored platelets which are currently disposed of after expiration for transfusion purposes.
The present invention is based on new knowledge discovered by the Inventors relating to molecular interactions between platelets and polymer fibers that be exploited for making beneficial compositions for treatment of wounds, achieving hemostasis, and implanting cells.
Citation or identification of any reference in Section 2 or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.